Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.

Table S1.Summary of regional changes in midguts of fed flies compared to midguts of starved flies.Related to the main Fig. 1.

Fig
Fig. S3.Related to the main Fig. 3. A-C) Heatmaps showing regional distribution of the progenitor cell cycle phases in G1 (A), S (B) and G2 (C) of the genotype esg-Gal4 ts >UAS-CycB-RFP, UAS-E2F1-GFP in starved and fed conditions.The number of G1 cells in A sums up ISCs and EBs since EBs possess only the G1 marker E2F1-GFP expression.Experimental design depicted in the main Fig. 2I.

Fig. S4 .
Fig. S4.Related to the main Fig. 4. A) Experimental design used to obtain data in the main Fig. 4C.Age matched, mated females of genotype esg-Gal4>UAS-CycB-RFP, UAS-E2F1-GFP, Delta-LacZ (Ctrl) in combination with Raptor-RNAi or Akt-RNAi, were aged for six days, and then shifted to the holidic diet for 3 days.B) Quantification of relative GFP+ cell number from the experiment depicted in the main Fig. 4D and E. Quantifications were performed from the R4-R5 regions from midguts of female flies of genotype esg ts F/O>UAS-GFP (Ctrl) in combination with Raptor-RNAi or Akt-RNAi.N guts are indicated in the figure panel.C) Quantification of Delta-LacZ signal intensity (α-β-Galactosidase immunostaining) from all α-Prospero positive EE cells from the experiment depicted in the main Fig. 4F.Measurements were from R1, R2, R4, R5 and borders flanking R3 from midguts of female flies kept in either starvation or holidic diet.Experimental design depicted in the main Fig. 1A.Pooled data from N starved =6 and N fed =5 midguts.N cells are indicated in the figure panel.P values in B were obtained by two-way ANOVA followed by Tukey's test.P values in C were obtained by Wilcoxon rank-sum test with multiple testing correction (FDR<0.05).

Fig. S5 .
Fig. S5.Related to the main Fig. 6 and 7. A) Experimental design used for ad libitum fed and intermittent fasted flies.Used to obtain data in the main Fig. 6 and 7. Age matched females of genotype esg-Gal4 ts >UAS-GFP, Delta-LacZ were kept at 29°C in the presence of males for 30 days in holidic diet (ad libitum fed) or flipped to starvation after three days feeding period (intermittent fasting).B) Quantification of total cell numbers from the experiment depicted in the main Fig. 6A.N guts , statistical test and significance are depicted in the figure panel.C) Total cell count comparisons along the midgut A/P axis between young (5d) and old (30d) ad libitum fed (left panel) and young (5d) and old (30d) intermittent fasted (right panel) female midguts.Light blue/gray and grey/red shadings are the standard deviation.P values in C were obtained by Wilcoxon rank-sum test using continuity and false discovery rate correction (FDR<0.05).

Table S2 . Adjusted p-values of mTOR regulator genes.
Related to the main Fig. 3K.